Proteome-wide and matrisome-specific alterations during human pancreas development and maturation.

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation.

Functional Bioinformatics Analyses of the Matrisome and Integrin Adhesome.

The extracellular matrix (ECM) is the noncellular compartment of living organisms and is formed of a complex network of cross-linked proteins, which is collectively known as the matrisome. Apart from providing the structure for an organism, cells interact and thereby communicate with the ECM. Cells interact with their surrounding ECM using cell-surface receptors, such as integrins. Upon integrin engagement with the ECM, cytoskeletal proteins are recruited to integrins and form a molecular protein complex known as the integrin adhesome. Global descriptions of the matrisome and integrin adhesome have been proposed using in silico bioinformatics approaches, as well as through biochemical enrichment of matrisome and adhesome fractions coupled with mass spectrometry-based proteomic analyses, providing inventories of their compositions in different contexts. Here, methods are described for the computational downstream analyses of matrisome and adhesome mass spectrometry datasets that are accessible to wet lab biologists, which include comparing datasets to in silico descriptions, generating interaction networks and performing functional ontological analyses.

Pathway and network analysis of genes related to osteoporosis.

As a common degenerative disease, osteoporosis (OS) is characterized by reduced bone mass and microarchitectural deterioration of bone tissue. Both genetic and environmental factors are involved in OS development. To date, ~300 genes have been confirmed to be involved in the pathogenesis of OS, a large majority of which have been independently investigated. As OS is a polygenetic disease, a comprehensive analysis focusing on the biological functions and interactions of OS­related genes would provide valuable information. In this study, OS related research deposited in PubMed was retrieved and genes related to OS were catalogued. Pathways with an enriched biological function for these genes were extracted, and the crosstalk between the enriched pathways was analyzed. A comprehensive network was constructed, and a minimal network was extracted using the Steiner minimal network algorithm. In this study, a total of 294 genes in were retrieved from PubMed. Biological processes found to be enriched included those related to bone metabolism and the immune system. In total, 58 pathways were enriched. Furthermore, the comprehensive network consisting of 3,943 nodes and 7,976 edges was constructed, among which 631 nodes and 2,581 edges contributed to the OS­specific molecular network. In this network, in excess of 300 potential genes associated with OS and two modules were identified. Thus, this study provides a mechanistic insight into OS and suggests more than 300 potential OS­related genes for future research.

Functional Insights from the Proteomic Inventory of Ovine Forestomach Matrix.

Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (dECM) biomaterial that serves as a scaffold for remodeling damaged soft tissue. dECM biomaterials are used in a variety of clinical applications, and their regenerative capacity is encoded not only in their biophysical properties but also in their molecular diversity. In this study, the proteome of OFM was characterized via both targeted and global mass spectrometry (MS) with the use of heavy isotope labeled (SIL) internal standards. Proteins were identified following either chemical digestion or extraction using saline or guanidine hydrochloride, followed by high resolution size exclusion chromatography. Identified proteins were annotated using the matrisome database and molecular function using the gene ontology database. The characterization identified 153 unique matrisome proteins, including 25 collagens, 58 glycoproteins, 12 proteoglycans, 13 ECM affiliated proteins, 20 ECM regulators, and 23 secreted factors. This inventory represents a comprehensive array of matrix proteins that are retained in OFM after processing. The diversity of proteins identified may contribute to OFM's remodeling capacity in clinical applications.

Modeling the Kinetics of Integrin Receptor Binding to Hepatic Extracellular Matrix Proteins.

The composition of the extracellular matrix (ECM) proteins and the expression of their cognate receptors dictate cell behavior and dynamics. In particular, the interactions of ECM proteins with integrin receptors are key mediators of these cellular processes, playing a crucial role in the progression of several diseases of the liver, including inflammation, fibrosis/cirrhosis and cancer. This study establishes a modeling approach combining computation and experiments to evaluate the kinetics of integrin receptor binding to hepatic ECM proteins. ECM ligand concentration was derived from LC-MS/MS quantification of the hepatic ECM from mice exposed to chronic carbon tetrachloride (CCl4); receptor density was derived from published literature. Mathematical models for ECM-integrin binding kinetics that were developed incorporate receptor divalence and an aggregation scheme to represent clustering. The computer simulations reproduced positive cooperativity in the receptor aggregation model when the aggregation equilibrium constant (Ka) was positive and greater than Keq for divalent complex formation. Importantly, the modeling projected an increase in integrin binding for several receptors for which signaling is known to be increased after CCl4 exposure in the liver. The proposed modeling approach may be of use to elucidate the kinetics of integrin receptor binding to ECM proteins for homeostatic and diseased livers.

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa.

The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called 'calcifying matrix' is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods).

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell.

Identification and Characterization of the Lysine-Rich Matrix Protein Family in Pinctada fucata: Indicative of Roles in Shell Formation.

Mantle can secret matrix proteins playing key roles in regulating the process of shell formation. The genes encoding lysine-rich matrix proteins (KRMPs) are one of the most highly expressed matrix genes in pearl oysters. However, the expression pattern of KRMPs is limited and the functions of them still remain unknown. In this study, we isolated and identified six new members of lysine-rich matrix proteins, rich in lysine, glycine and tyrosine, and all of them are basic matrix proteins. Combined with four members of the KRMPs previously reported, all these proteins can be divided into three subclasses according to the results of phylogenetic analyses: KRMP1-3 belong to subclass KPI, KRMP4-5 belong to KPII, and KRMP6-10 belong to KPIII. Three subcategories of lysine-rich matrix proteins are highly expressed in the D-phase, the larvae and adult mantle. Lysine-rich matrix proteins are involved in the shell repairing process and associated with the formation of the shell and pearl. What's more, they can cause abnormal shell growth after RNA interference. In detail, KPI subgroup was critical for the beginning formation of the prismatic layer; both KPII and KPIII subgroups participated in the formation of prismatic layer and nacreous layer. Compared with different temperatures and salinity stimulation treatments, the influence of changes in pH on KRMPs gene expression was the greatest. Recombinant KRMP7 significantly inhibited CaCO3 precipitation, changed the morphology of calcite, and inhibited the growth of aragonite in vitro. Our results are beneficial to understand the functions of the KRMP genes during shell formation.

Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation.

The extracellular matrix (ECM) of the brain plays crucial roles during the development, maturation, and regeneration of the CNS. In a subpopulation of neurons, the ECM condenses to superstructures called perineuronal nets (PNNs) that surround synapses. Camillo Golgi described PNNs a century ago, yet their biological functions remain elusive. Here, we studied a mouse mutant that lacks four ECM components highly enriched in the developing brain: the glycoproteins tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans brevican and neurocan. Primary embryonic hippocampal neurons and astrocytes were cultivated using a cell insert system that allows for co-culture of distinct cell populations in the absence of direct membrane contacts. The wild-type and knock-out cells were combined in the four possible permutations. Using this approach, neurons cultivated in the presence of mutant astrocytes displayed a transient increase of synapses after 2 weeks. However, after a period of 3 weeks or longer, synapse formation and stabilization were compromised when either neuron or astrocyte cell populations or both were of mutant origin. The development of PNN structures was observed, but their size was substantially reduced on knock-out neurons. The synaptic activity of both wild-type and knock-out neurons was monitored using whole-cell patch clamping. The salient observation was a reduced frequency of IPSCs and EPSCs, whereas the amplitudes were not modified. Remarkably, the knock-out neuron phenotypes could not be rescued by wild-type astrocytes. We conclude that the elimination of four ECM genes compromises neuronal function.

A comprehensive protein expression profile of extracellular matrix biomaterial derived from porcine urinary bladder.

AIMS: To generate a comprehensive profile of the protein composition of xenogeneic biomaterial, derived from porcine urinary bladder matrix (UBM). MATERIALS & METHODS: Tunica layers and muscularis mucosa were removed from bladders using mechanical delamination. UBM was prepared using a solution of peracetic acid in ethanol, lyophilized then milled into powder. UBM biomaterial was subjected to tryptic digests and components separated using high-performance liquid chromatography with an ion trap mass spectrometer and identified through databases. RESULTS: A repertoire of 129 proteins with neurotrophic, antiangiogenic and tumor-suppressive activities and those associated with tissue remodeling and wound repair were identified. CONCLUSION: This study provides the first insight into the complex nature of the UBM and how its application may be tailored for specific applications in regenerative medicine. We propose that the UBM be further investigated for reconstructive and regenerative remodeling of cardiac and dermal tissues, as well as peripheral nerves.

Podocan-like protein: a novel small leucine-rich repeat matrix protein in bone.

Recently, significant attention has been drawn to the biology of small leucine-rich repeat proteoglycans (SLRPs) due to their multiple functionalities in various cell types and tissues. Here, we characterize a novel SLRP member, "Podocan-like (Podnl) protein" identified by a bioinformatics approach. The Podnl protein has a signal peptide, a unique cysteine-rich N-terminal cluster, 21 leucine-rich repeat (LRR) motifs, and one putative N-glycosylation site. This protein is structurally similar to podocan in SLRPs. The gene was highly expressed in mineralized tissues and in osteoblastic cells and the high expression level was observed at and after matrix mineralization in vitro. Podnl was enriched in newly formed bones based on immunohistochemical analysis. When Podnl was transfected into osteoblastic cells, the protein with N-glycosylation was detected mainly in the cultured medium, indicating that Podnl is a secreted N-glycosylated protein. The endogenous Podnl protein was also present in bone matrix. These data provide a new insight into our understanding of the emerging SLRP functions in bone formation.

Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.

Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.

Serum markers of liver fibrosis: combining the BIPED classification and the neo-epitope approach in the development of new biomarkers.

BACKGROUND: Fibrosis is a central histological feature of chronic liver diseases and is characterized by the accumulation and reorganization of the extracellular matrix. The gold standard for assessment of fibrosis is histological evaluation of a percutaneous liver biopsy. Albeit a considerable effort have been invested in finding alternative non-invasive approaches, these have not been sufficiently successful to replace biopsy assessment. AIM: To identify the extracellular matrix proteins of interest, that as protein degradation fragments produced during extracellular matrix metabolism neo-epitopes, may be targeted for novel biochemical marker development in fibrosis. We used the recently proposed BIPED system (Burden of disease, Investigative, Prognostic, Efficacy and Diagnostic) to characterise present serological markers. METHODS: Pubmed was search for keywords; Liver fibrosis, neo-epitopes, biomarkers, clinical trail, extra cellular matrix, protease, degradation, fragment. RESULTS AND CONCLUSION: Implementation of BIPED categorization in the development and validation of fibrosis biomarkers to simplify and standardize the use of existing and future biomarkers seems advantageous. In addition, a systematic use of the neo-epitope approach, i.e. the quantification of peptide epitopes generated from enzymatic cleavage of proteins during extracellular remodeling, may prove productive in the quest to find new markers of liver fibrosis.

hOLFML1, a novel secreted glycoprotein, enhances the proliferation of human cancer cell lines in vitro.

We describe a novel secreted protein, named hOLFML1 (human olfactomedin-like protein 1), with an olfectamine domain in its C-terminus, mainly expressed in the small intestine, liver, lung and heart. Immunohistochemical staining on human small intestine indicated that the protein localizes preferentially in the intestinal villi. Interestingly, ectopic hOLFML1 promoted proliferation of HeLa cells and increased the percentage of cells in S phase. In contrast, knock down of hOLFML1 protein expression by siRNA inhibited cell proliferation and delayed the entry of cells into S phase. Our data also revealed that hOLFML1 is N-glycosylated and its secretion is triggered by serum. Taken together, these findings suggest that hOLFML1 may play a significant role in the regulation of cell proliferation in vitro.

Essential requirement for zebrafish anosmin-1a in the migration of the posterior lateral line primordium.

Kallmann syndrome (KS) is a human genetic disease that impairs both cell migration and axon elongation. The KAL-1 gene underlying the X-linked form of KS, encodes an extracellular matrix protein, anosmin-1, which mediates cell adhesion and axon growth and guidance in vitro. We investigated the requirement for kal1a and kal1b, the two orthologues of the KAL-1 gene in zebrafish, in the journey of the posterior lateral line primordium (PLLP). First, we established that while the accumulation of kal1a and kal1b transcripts was restricted to the posterior region of the migrating primordium and newly deposited neuromasts, the encoded proteins, anosmin-1a and anosmin-1b, respectively, were accumulated in the PLLP, in differentiated neuromasts and in a thin strip extending along the trail path of the PLLP. We also show that morpholino knockdown of kal1a, but not kal1b, severely impairs PLLP migration. However, while the PLLP of kal1a morphants displays highly abnormal morphology, proper expression of the cxcr4b gene suggests that kal1a does not play a role in PLLP differentiation. Conversely, wild-type levels of kal1a transcripts are detected in the PLLP of cxcr4b or sdf1a morphant embryos, strongly suggesting that kal1a transcription is independent of CXCR4b/SDF1a signalling. Last, moderate depletion of both anosmin-1a and SDF1a markedly affects PLLP migration providing strong evidence that anosmin-1a acts as an essential co-factor in SDF1a-mediated signalling pathways. Our findings, which demonstrate, for the first time, an essential requirement for anosmin-1a in PLLP migration, also strongly suggest that this protein plays a key role for proper activation of the CXCR4b/SDF1a and/or CXCR7/SDF1a signalling pathway in PLLP migration.

Extracellular SH3 domain containing proteins--features of a new protein family.

In the year 1994, the protein MIA (melanoma inhibitory activity) was found to be strongly expressed and secreted by malignant melanomas and subsequent studies revealed that MIA has an important function in melanoma development and invasion. Multidimensional NMR-spectroscopy and x-ray crystallography revealed that recombinant human MIA adopts a Src homology 3 (SH3) domain-like fold in solution, a structure with two perpendicular antiparallel three- and five-stranded beta-sheets. SH3 domains are protein modules that are found in many intracellular signalling proteins and mediate protein-protein interactions by binding to proline-rich peptide sequences. Unlike previously described protein structures with SH3 domain folds, MIA is a secreted single-domain protein of 12 kDa that contains an additional antiparallel beta-sheet and two disulfide bonds. Furthermore, the charge surrounding the canonical binding site differs from that of classical SH3 domains. The two disulfide bonds are crucial for correct folding and function as revealed by mutation analysis. Therefore, MIA appears to be the first extracellular protein adopting an SH3 domain-like fold. MIA was shown to interact with fibronectin, and MIA-interacting peptide ligands identified by phage display screening are similar to the consensus sequence of type III human fibronectin repeats, especially FN14. Interestingly, recent data revealed that MIA can also directly bind to integrin alpha 4 beta 1 and alpha 5 beta1 and that it modulates integrin activity negatively. These findings suggest an interesting role of the SH3-domain proteins in the extracellular compartment. Recently, MIA homologous proteins with a sequence identity of 44% and a sequence homology of approximately 80% were determined (TANGO, MIA-2, OTOR). This clearly suggests that this structural device is used more frequently, in processes ranging from developmental changes to the interference of cell attachment in the extracellular matrix. Detailed studies are necessary to determine the exact function of the MIA homologous proteins. It will be interesting to know whether additional protein families can be identified which are secreted and carry SH3 domain-like modules, in addition to elucidate what the specific physiological targets of this protein family are.

Elucidation of subfamily segregation and intramolecular coevolution of the olfactomedin-like proteins by comprehensive phylogenetic analysis and gene expression pattern assessment.

The categorization of genes by structural distinctions relevant to biological characteristics is very important for understanding of gene functions and predicting functional implications of uncharacterized genes. It was absolutely necessary to deploy an effective and efficient strategy to deal with the complexity of the large olfactomedin-like (OLF) gene family sharing sequence similarity but playing diversified roles in many important biological processes, as the simple highest-hit homology analysis gave incomprehensive results and led to inappropriate annotation for some uncharacterized OLF members. In light of evolutionary information that may facilitate the classification of the OLF family and proper association of novel OLF genes with characterized homologs, we performed phylogenetic analysis on all 116 OLF proteins currently available, including two novel members cloned by our group. The OLF family segregated into seven subfamilies and members with similar domain compositions or functional properties all fell into relevant subfamilies. Furthermore, our Northern blot analysis and previous studies revealed that the typical human OLF members in each subfamily exhibited tissue-specific expression patterns, which in turn supported the segregation of the OLF subfamilies with functional divergence. Interestingly, the phylogenetic tree topology for the OLF domains alone was almost identical with that of the full-length tree representing the unique phylogenetic feature of full-length OLF proteins and their particular domain compositions. Moreover, each of the major functional domains of OLF proteins kept the same phylogenetic feature in defining similar topology of the tree. It indicates that the OLF domain and the various domains in flanking non-OLF regions have coevolved and are likely to be functionally interdependent. Expanded by a plausible gene duplication and domain couplings scenario, the OLF family comprises seven evolutionarily and functionally distinct subfamilies, in which each member shares similar structural and functional characteristics including the composition of coevolved and interdependent domains. The phylogenetically classified and preliminarily assessed subfamily framework may greatly facilitate the studying on the OLF proteins. Furthermore, it also demonstrated a feasible and reliable strategy to categorize novel genes and predict the functional implications of uncharacterized proteins based on the comprehensive phylogenetic classification of the subfamilies and their relevance to preliminary functional characteristics.

Expression and characterization of disulfide bond use of oligomerized A2-Pancortins: extracellular matrix constituents in the developing brain.

The region-specific characteristics of the extracellular matrix are crucial for diverse functions in the brain. Pancortins/neuron-specific olfactomedin-related glycoproteins are components of the extracellular matrix. They comprise four alternatively spliced variants, Pancortin-1 to -4, which share a common portion, the B part, in the middle of their structure, have two pairs of alternatively spliced 5' regions, A1 and A2, and 3' regions, C1 and C2. Here we demonstrate that in mice, Pancortin-3 (A2-B-C1) and Pancortin-4 (A2-B-C2), which we have grouped together the A2-Pancortins, were transcribed early during the development of the brain in a region specific manner and were expressed very stably in vivo. They are N-glycosylated and secreted. Furthermore, we examined their ontogenetical expression profiles in the developing thalamus using antiserum against the common B region, since transient expressions of their mRNAs were notable there. In the developing thalami, they lasted long in oligomerized form even after the transcription of their mRNAs decreased to an undetectable level. Further analyses revealed that cysteine residues that are located in the common B part are important for homo- and hetero-oligomer formation of A2-Pancortins. When we substituted cysteine residues 45 and 47 with serine residues in that common B part, oligomerization of the A2-Pancortins was highly disturbed.

hOLF44, a secreted glycoprotein with distinct expression pattern, belongs to an uncharacterized olfactomedin-like subfamily newly identified by phylogenetic analysis.

Secreted proteins are indispensable for the development and differentiation of multicellular organisms. Cloning and characterization of novel or hypothetical genes encoding these proteins are therefore inviting great incentives. Using bioinformatics tools and experimental approaches, we isolated and characterized a human secreted glycoprotein, hOLF44, which contains a highly conserved olfactomedin-like (OLF) domain in the C-terminal. However, phylogenetic analysis revealed that hOLF44 is not clustered into any of the OLF subfamilies containing characterized members, and obviously falls into a newly identified uncharacterized OLF subfamily. Western blot analysis showed that hOLF44 protein is robustly secreted from the transfected COS-7 cells. Expression levels of hOLF44 mRNA are abundant in placenta, moderate in liver and heart, whereas fairly weak in other tissues examined. Immunohistochemical study on human term placenta demonstrated that hOLF44 is mainly localized extracellularly surrounding the syncytiotrophoblastic cells and very rarely expressed in the maternal decidua layer. These results suggest that hOLF44 may have matrix-related function involved in human placental and embryonic development, or play a similar role in other physiological processes. The further functional characterization of hOLF44 may provide insights into a better understanding of the newly identified OLF subfamily.